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EJtrogcnic Activity of DDT Analogs and Polychlorinated Biphenyls Joel Bitman* and Helene C. Cecil Because of (he geometric similarity of DOT to the synthetic estrogen, stilbestrol, DDT and 52 related compounds were tested in a sensitive estrogen assay in rats. Estrogenic activity was evaluated using the I fl-hr glycogen response of the immature rat uterus. Diphenylmethane, diphenylethane, and triphenylmcihane compounds were active when a p* or a'-posi tion was unoccupied or occupied by an hydroxy or methoxy group. Halida or alkyl groups in the p,p'positions rendered the compounds inactive. Poly chlorinated biphenyls and polychlorinated tri- fihenyls, compounds which are environmental poi- utants of industrial origin, were cstrogcnkally active. Phenoiphthalol and phenolphthaiein, com pounds which are used as laxatives in drug prepara tions, were also estrogenically active since they con tain tha appropriate p,*'-dihydroxy structures. Stereo models indicated tnat p,p'-dihydroxy com pounds of the active nuclei would have internudear distances of the hydroxyl groups which would ap proximate those of the natural steroidal estrogens and the synthetic stilbene estrogens. n 1945 Solmssen published an excellent and comprehen (p-chlorophenyl)eihylene; p,p'-DDMU, I-chk>ro-2,2-bts- I sive 117-page review of the synthetic estrogens and the relation between their structure and activity. In the (p-chlorophenyl)elhylene; and p.p'-DDA, 2,2-bis(^chiorophenyi)acetic acid. 25 years since Sotmssen's review, DDT, a chlorinated hydro Table II: o.p'-DDT, l,l,l-trich!orc^2-(p-chlocophenyl)-2- carbon with a geometric similarity to (he synthetic estrogens, (o-chlorophenyl)ethane; o.p'-DDE, l.l-dkhloro^-fp-chloro- has been widely used throughout the world for pest control. phenyl),2-<0-chiorophenyi)ethylene; o.p'-DDMU. 1-chloro- The recent demonstration by Welch et al. (1969) of the estro 2-(p-chlorophenyl), 2-(o-chlorophenyl)ethylene; >;.p'-DDD, genic activity of o,/'-DDT and our own investigations (Bitman et /., 1966) have prompted us to investigate a series of DDT analogs, homologs, and structurally related compounds 1.1 -dichloro-2-(p-chlorophenyl),2-(0-chlorophenyl)ethane; w.p'-DDD, l,l-dichloro-2><p-chlorophenyt),2-(/n-chloro- phenyl)ethane and p.p'-Methoxychior. l.t.Mrichloro-?,2-bii- in an attempt to determine relationships of structure to estro (p-methoxyphenyl)ethane. genic activity. Table Uf: Compound 20, 1,1-Diphenylmethane; 21, 1.1- METHODS Dichloro-1,1-diphenylmethane; 22, 1,1 bMp-bromophenyl)methane; 23, l-(phenyl)-l-(p-ltydroxyphcityi)mcthene; 24, We used the sensitive 18-hr glycogen response of the rat l,I-bis(p-hydroxyphenyl)methane; 25, Mp-hydroxyphenyl)- uterus as a measure of estrogenic activity (Bitman et a/., Mp-methoxyphenyi)melhane; 26, WphenylM-lp-methovy- 1965). The potency of active compounds is reported in o-hydroxyphenyl)meihane; 27, 1,1 -bis(o-hydroxyphenyl)- terms of the minimal subcutaneous dose which will increase methane; 28, l.l-biko-hydroxy-m-cMorophenyljmethane; glycogen to a level significantly different from control. The 29, Hphenyl)-Hp<htorophenyQ-l-methanol; 30. 1,1-bistp- IS-hr glycogenic response is illustrated in Figure 1, in which chlorophenyl)-!-methanol; 31, 2,2'-dihydroxybenzophenone; the dose-response curve for o.p'-DDT is represented. The 32, 2.4-dihydroxvbeniophenone; 33, 4t4'-dihydroxybenztv steeper response line for glycogen, as compared to uterine phenone; and 34, 2,2'-dihydroxy-4.4'-dimeihoxybenxophe- weight, is readily apparent. none. Test substances were dissolved in olive oil or an aqueous Sources of the compounds used in this study were: Noe. ethanol solution and injected subcutaneously at a screening 4, S, 16--Rohm and Haas, Philadelphia; 9, 13--Dr. C. F. dose rate of 8 mg per rat. Immature female Witter rats Fries, U.S. Dept. Agr., Beltsville; 17--E. K. Du Pont de (21-23 days old; 36-48 g) were killed 18 hr after the injection; Nemours & Co., Inc., Wilmington; 18--Sigma Chemical Co., uteri were quickly excised, weighed, and analysed for glycogen St. Louis; 24, 27, 35, 42--K A K. Laboratories, (nc., Ptam- by the enthrone procedure (Seifter et a/., 1950). Substances view, N.Y.; 38--Eastman Kodak Co., Rochester; 43-33-- showing activity were tested further at dosage levels to 0.05 Monsanto Co.. St. Louis. All other compounds were pur mg. Statistical comparisons were made using Student's t test chased from the Aldrich Chemical Co., Ine., Milwaukee. with correction for unequal group siae. Purity, as given by the manufacturers, was better than 99%. ABBREVIATIONS TitMe I: p.p'-DDT, 1.1,1 -trichkjro-2j2-bis(p-chlorophcnyQelhane; Tetrachloro-DDT, l,l,l,2rtetrachloro-2,2-bis(p-chtorophenyDethane; p,p'-DFDT, l,ltl-trichloro-2J-bis(/>>fluorophenyl)ethane; p.p'-Penhane, l,l,l-trichloro-2,2Msfp-ethylphenyltethaite; p./t'-Keithsm, 1,1,] -trkhloro-2,2bis(p-chlorophcnyl)ethane; p,p'-DDTF, l,l,l-trif!uoro-2Jbis(p>chloropfcenyt)etham{ p.p'-DDD, l,l-dteMoro-2^l-bit(p-chlorophenyOethane; p.p'-DDE, I,I-dkhloro-2^-bis- Animal Husbandry Research Division, BeHsviile, Md. 20705 * To whom correspondence should be addressed. RESULTS AND DISCUSSION The natural estrogens are steroids which contain a phenolic ring A and an oxygen function at the C, position, white the synthetic estrogens, which are stilbene derivatives, contain two phenolic rings (Figure 2). It is apparent that active estrogenicity is dependent upon the presence of at least on* phenolic hydroxy ring structure. In most estrogen tests these compounds are active in the mkrogram or sufaancrovam range. v In contrast to this, the chlorinated hydrocarbon pesticides related to DDT are only active as estrogens in millipam amounts, a KXXWfoki difference. The DDT analogs are not phenolic, but they may give rist to aromatk phenolic sub stitution during metabolic conversions in the animal. The llOt 1. AOX, rooo CHIM., VOL. II, NO. . 1,7, j-i. I, MQNS 087130 Of`DOT Plgere t. Dmmmpmm rtklkwNpt iMm wsight, flycr>*a, Mi r,/'-DDT DOT analogs are compounds of Ihe diphenylethane type (Figure 2). Other analogs tested were compounds of Ihe diphcnytmcihane or triphenylmethane series. We have also examined polychlorinated biphenyls and polychlorinated triphcnylt, compounds which have become increasingly im plicated as environmental pollutants of industrial origin. Wc have not included in this study any steroids, synthetic estrogens, or anti-estrogens of the stilbene structure, and have excluded almost all compounds of the coumarin, isoflavone, anthracene, and phenanthrene type. DIPHBNYLBTHANB COMPOUNDS p,p'-PeaMaoa Occupied by Halida or Alkyl, The com pounds evaluated in Table I are diphenylethane derivatives in Table I. Dlpbewytemawa Compoaada Nb pj'-Padtitm Occupied by HaHdt ar Alkyl Graupp R R' No. X R It' NflOM 1a 2 ci h --CCIi p.p'-DDT . a --CO* Tetrechtoro-DOT 3F H -CCI. p.p'-DFDT 4 CHiCHt H --CCli p,p'-Pertfiane 3 Cl OH --CO* p,p'-Kehhane 6 Cl H --CP* p.p-OOTP 7 Cl H --CHClt p,p'-DDD sa pa io a ... ... --^^eCwla pe.,ap''--DDODEMU h --COOH Af'-DDA . * M.B.D. mkimwN *ftedv* 4am. 11 - insert**. Actfdty M.E.O.* ** 4 4 l* I* I* I* l* I> P I* which the p.p'-positiens are occupied by halide or alkyl groups. Almost all were dpvotd of estrogenic activity; OOT (empd I) and tctrachloro-DDT (empd 2) exhibited a slight glycogenic response. It appears that halide or alkyl substitutions in the /,//-positrons were stable, and during me tabolism in the animal body, little if any p^'-phenolic hydroxy compounds are produced. /vorp.p' Position Occupied by --Her--OCH*. When one of the para positions of the aromatic ring is substituted by a hy drogen or methoxy group, the compound exhibits estrogenic activity (Table II). Potency is of a low order of magnitude, being approximately 1000 times less active than compounds of the stilbene series, but n similar in potency lo coumarin and isoflavone estrogens (Bickoff cr a/., 1960). The minimum effective dose (MED) of diethylstilbestrol which elicited t glycogen response was 0.1 pg, as compared lo Ihe most active compound of Table II, o.p'-DDT, empd I), whose MED was 0.23 mg. The phenolic character of the natural and synthetic estro gens has demonstrated the dependency of estrogenicity upon the presence of a phenolic structure. The aromatic rings of the active compounds of Table II are open, i.e., they have a p or p'-position occupied by --H and may give rise to phenolic substitution during metabolism. There also appears to be a requirement for the ethane chain to be inert, it, either the trchloroethane (-CH-CCI*) or the vinyl halide group (>C CCU) mutt also be present (empds 11,12,13). Thus, empda 14, 13, and 19, containing more reactive 2-carbon chain con figurations, are inactive, even though one of the aromatic cinp could be hydroxylated to the phenolic structure. W hove concluded that rapid in ofee metabolism of these compounds ia responsible for their tack of activity. In the stilbestroi series(Sotmseen, 1943) and in thecoumarin series (Btckoff tt d., I960) of estrogsna, p,p'-dim*thoxy con* pounds are less active than comparable p^'-dihydvoxy com pounds. In the chlorinated diphenylethan* win (Table II), the p^'-methoxy compound, methonycMor (empd 17) woe *O NS 0 8 7 1 3 1 J.L.M >. AO*. POOD CHIMh VOL. It. NO, l I,m MO* nMtn. Cmitmit*i>-mi>'-PmtlkmOKWik4*r-Hor-OCH. Ne. P p' o 11 H -- a 12 H 1) H a a 14 H a IS H a aa a a wQ 16 H HH 17 OCH, OCH. H It OCM* OCH. H 19 H H '-Cl R H H H H H H H ' M.B-D. - minimum Ufkctive Com. * 1I - inactive. R' R' --CC1, --ca. --CHO --CKO. --CHO. --ca, --ca. --ca. --CHO Neew *p'-DDT e.p'-DDB o.p'-DDMU o.p'-DDD m.p'-DDD l,M>Trichioro>2,2'bMphenyl)erhetw p.p'-Methoxychiov Tech. Methoxychlor -4- e.p'-) 2,2>Bls(e-chlorophsnyl)ecetaldshyde m 0.25 4 1 f 1* 1 4 1 l approximately at active as other compounds which might give rise to phenotk hydroxy substitution on metabolism. Tech nical methoxychlor (empd 18), which may contain an o,p'methoxychlor, was four times more active than pure methoxychlor. A series of dipheitylmethane compounds was examined to determine structural correlates of estrogenic activity (Table III). Active compounds contained either one or two ^hy droxy or p-methoxy groups (empds 23, 24, 25, 32, 33, 34). TaMa The most active compound was p.p'-dihydroxydiphenylmethane which elicited a glycogenic response el the I mg dose level. Solmssen (1945) reported activity for this compound at the 100 mg level, but the differences in bioessay procedures could explain part of this difference in result, (n the diphenylethene series, compounds with a p-hydrogen and a stable ethane chain were metabolized to active estrogens, probably containing a p-hydraxy structure. In contrast, diphenylmethane compounds with a p-hydrogwi ware not active, probably being metabolized rapidly at the methane linkage and excreted from the body. Benzophenone derivatives, which contain the more stable ketone structure at the methane carbon, were active if a p-hydroxy was present (Table III). Ne, P P' R TT 1Diphwtylmelhena derivative1 30 H 21 H 22 Br 23 H 24 Oft 25 OCM* 28 OCH, 27* H 21' H 29 H 30 a 31 H H H Br OH OH OH H H H a a H HH aa HH HH H H xH H H HH HH H OH H OH P I* P 2 1 4 P P P P P P BsneophsnenedwtvedvM 32 H 33 ' OH 34 OCH, OH OH OCH, 2 2 4 M.B.D. jMj<why* * bM-*yUrec:r, m dUorogbrnyOm Two diphenylpropane compounds were active (empds 35 and 36), the p.p'-hydrmy compound exhibiting much greater activity than a p,p'dimethoxy compound. Dihydroxy di* phenyl propane (empd 33) wessi active as c,p'-DDT. Sdms- sen (1945) found that this compound was active at a 100 mg Since the p,p'-dihydroxy structure appeared to be the struc ture conferring activity, phenolpbthelol, a phenyl substituted diphenylmethane compound containing p.p'-dihydroxy groups was tested. PhenolphtheId was as potent as any compound of the types studied. Ring closure, as in phenoJphthatetn, resulted in a 20-fold loss in potency. These compounds are not known to have estrogenic activity and art extensively used as laxatives in a number of drug preparations. In Table IV two miscellaneous derivatives which bear some relation to closed ring diphenylmethans structures are in cluded: fluorene and 9,10-dimethylanthrecent. Both of thesecompounds were Inactive when tested at dost levels up to 8 mg per ret. BIPHENYL AND TRIPHBNYL COMPOUNDS ' the 4 end 8 mg dose levels (Table V). In a series of poly chlorinated biphenyls, the compoands containing up to 48% 111# I. AOR. POOD CMBMw VOL. IA NO. A * MONS 087132 JOCT Q~Oi M-H: liraams task tut srrtNNe rk i>.ik DirtWNVt IfMAMS 0-0: M-H: *4k io.rt -c-p- DtFHSMVtMCrHAtW -'''hi Ml MPHIMTIPROPAMt CH-* TSteHINHMIMAMV tpHttm TIlfWINYt 0-0: AtoI .*X H-H: teak Arol u.al ts.ok 0-0: A roc ask M-h: rak AtoC *sl ml Ofm L Strirtwl fow--lee tf cstecewdc tampawm+ these products are crude mixtures containing a number of compounds. A polychlorinated triphenyl containing 42% chlorine was found to be more active, at a I mg dose level. Schueler (1946) and his coworken (Fisher et a/., 1952; Keasling and Schueler, 1950) have theorised that a rather large, rigid, lipoid soluble molecular structure with two active hydrogen-bond forming groups located at an optimum dis tance of 14.5 A units from each other would be estrogenic. They further stated that potency is decreased as the distance between groups is decreased or increased. While DOT possesses e relatively large, rigid, lipoid soluble molecular constitution, it does not present active hydrogen toms at the hypothesised optimum distance of 14.5 A, how ever. The presence of the electronegative chlorine atoms in the ^'-orientations would prohibit the existence of active hydrogen. If these pjt` chlorine atoms were tmtabjUeud to groups possessing active hydrogen, the poasibiHty of estrogen action would exist The gsnaral lack of estrogenic activity of p.p'-DDT analop suggests that such metabolism does not occur readily in the biological situations studied thus far. Conversely, the activity of o^'-DDT raises interesting theoretical rdettaaMps between chemical constitution and estrogenic activity. The chlorine atoms are not at the hypothesised optimum distance. The exact nature of the active estrogen structure arising from o.p'-DDT, if it is not o,/s'-DDT itself, might provide important information relating to the spatial configuration of an active estrogen. Drefcflng Stereomodels were constructed of many of the active structures to determine whether consistent stereochemi cal factors were prsism. Intemudsar distancee were mea sured Mid were given in Figure 2 for both O to O atoms of assumed dihydroxy compounds, and for the H to H distance of the hydroxyl group. Interatomic distances for the DreidIng medals of esteem and rtilbene were found to be much Table IV. Dlpheaylpcepaas and Tripbewrhurthane Campounds ActHMy M.tD.* Ne. 35 mo<0>_|~<0>oh 2.2-BtUx-hy- 0.23 <! , droxyptun- m W1 '"."-S-OoCM, 1.3-Dichloro- 4 2.2-bid^ & plMnyl)- :h,oh "Cue phthalstn 9,10-Dtnmh- t * M.B.D. - mialwem affective dess. I inactive. i j.t. i I. AOR. FOOD CHIM, VOL. II, NO. A 1*7, till TaMa V. Mpbnyt and Tripbnyl CMpadi @H> N Name 41 o,o'>Biphenol 42 p.p'.Biphenol 2.2'Dihydrtmydlphanyl 4,4 '-DihydfOKyihpbwtyl Polychlorinated Biphanyl(PCB). 43 PCI Aroelor 1221 217, Chlorine 44 PCB Arodor 1232 32% Cl 45 PCB Arodor 1242 4255 a 49 PCB Arodor I24g 4i% a 47 PCB Aroelor 1234 34% Cl 41 PCB Arodor 1290 60% Q 49 PCB Arodor 1292 92% a " 30 PCB Arodor I29f M%a 31 PCB Arodor 4463 60% PCB. 40% polychlori nated triphanyl (PCT), 63% a 32 PCT Arodor 3442 42% a 3) PCT Arodor 3460 60% a 1 M.B.D. - minimum effective dote. * 1 - inactive. m 4 a i 9 t 9 1* p p p p 1 p amailer than the 14.5 A quoted by Keasling and Schueler (1950). The 10.9 A we round agree closely with the X-ray crystallographic data of Norton el at. (1963,1964), who found 10.93 A for 170-eatradM. This discrepancy in interatomic dif ferences may be related to the improved accuracy of the cur rent atomic models, when compared to those used in 1950. The diphenyleihane, diphenylmethene, dlphenylpropane, trlphenybnethane, biphenyl, and triphenyl compounds all have interatomic distances of 9.4 to 10.3 A for the moat Ukely O to O substitutions. The H to H internuckar distances ofthe hydroxyl group* range from 9.1 to tl.l A in these compounds Both the O--O and H--H intermiciear distances; therefore. arm only slightly smaller then corresponding bond dbumis in natural and synthetic estropsna. The structural observations rrgerrllng estrogsrUc activity in the compounds studied Infested that activity is conferred when e p- orp'-poeition is unoccupied (--H), or is substituted by --OH or --OCH. Halide, or alkyl groups, occupying the A stable ethane chain was found to be nccesary for activity, t.g., the trkhioroethine or the inert vinyl haNde group; if either C of the ethane chain bears an oxygen function (alcch hoi, aldehyde, or acid), the compound is metaboiiad and no estrogenic activity is observed. Some polychlorinated bi phenyl and triphenyl compounds exhibited estrogenic activity. Measurements of. internudear distances of Dreiding sterie models indicated that active sitea would be 9-11 A span, a range similar to those found in natural and synthetic estro gens. Quantitatively similar estrogenic activity was obtained with a series of diphenylmethene or triphenytmelhana deriva tives which contained p*OH functions. Correlations of struc ture with activity suggest that the active eetrogme derived from o.p'-anelogs of DOT are o-phenotic metabolites. LITERATURE CITED Bkkoff, E. M., Livmgalon, A. L., Book, A. N.. Arch. Btaehrm. 10, 262 (1990). Btimen. 1., Cecil, H. C, Hants, S. J.. Fries, Q. F . Srknce tag, 371 (1961). Bitnwn, /,, Cecil, H. C.. Mench, M. L.. Wreim, T. R., Badacm otogr 79.63(1963). FIher, A. L, Keeabng, H. H., Schueler, F. W,, Pme. Sat. Cep. Bta. Mad. 11,439(1932). KeasUng.H. H., Schueler, F. WAmir, Pham. As*. 99,17(1939k Norton, D. A., Karthe. O , Lu. C. T,, Acta Cry*. 19,19(1993). Norton, D. A.. Kartha. O., Lu. C. T.. Acta Crytt. 17,77(1964), Schueler, P. W , Scitmw ttt, 211 (1946). Setter, 9., Dayton, S., Novie, g., Muntwyler, B., Are*. Bhctmm. g, 191 (1950). Srin--en. U. V.. Cham. Baa. St, 411 (1943). Welch. R. M., Levin, W., Cowwy, A. H., Taxkat. Appt. Hamm*. 1059(1969). Mctitim/for mirw Jam 33,1970. .icccptmt Aagaat 13,1979. \ 1IU I. AOR. FOOD CHIM.. VOL. IB, NO. *, 1970 \ MONS 007134